Activated protein c variants with normal cytoprotective activity but reduced anticoagulant activity

ABSTRACT

Variants (mutants) of recombinant activated protein C (APC) or recombinant protein C (prodrug, capable of being converted to APC) that have substantial reductions in anticoagulant activity but that retain normal levels of anti-apoptotic activity are provided. Two examples of such recombinant APC mutants are KKK191-193AAA-APC and RR229/230AA-APC. APC variants and prodrugs of the invention have the desirable property of being cytoprotective (anti-apoptotic effects), while having significantly reduced risk of bleeding. The invention also provides a method of using the APC variants or prodrugs of the invention to treat subjects who will benefit from APC&#39;s cytoprotective activities that are independent of APC&#39;s anticoagulant activity. These subjects include patients at risk of damage to blood vessels or tissue in various organs caused, at least in part, by apopotosis. At risk patients include, for example, those suffering (severe) sepsis, ischemia/reperfusion injury, ischemic stroke, acute myocardial infarction, acute or chronic neurodegenerative diseases, or those undergoing organ transplantation or chemotherapy, among other conditions. Methods of screening for variants of recombinant protein C or APC that are useful in accordance with the invention are also provided.

This is a continuation of co-pending application Ser. No. 10/886,766,filed Jul. 8, 2004, to which priority under 35 U.S.C. §120 is claimed,and which claims the benefit of provisional application Ser. No.60/485,792, filed Jul. 8, 2003, under 35 U.S.C §119(e), all the entiredisclosures of the prior applications are incorporated herein byreference.

This invention was made with the U.S. Government support under ContractNo. HL52246 by the National Institutes of Health. The U.S. Governmenthas certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to variants (mutants) of recombinantprotein C and activated protein C, an enzyme that normally hasanti-thrombotic, anti-inflammatory, and anti-apoptotic activities. Therecombinant activated protein C mutants of the invention have markedlyreduced anticoagulant activity, but retain near normal anti-apoptotic(cytoprotective) activity, so that the ratio of anti-apoptotic toanticoagulant activity is greater in the variants than it is inwild-type or endogenous activated protein C. This invention also relatesto methods of using these variants. The activated protein C variants ofthe invention are useful as inhibitors of apoptosis or cell death and/oras cell survival factors, especially for cells or tissues of the nervoussystem, which are stressed or injured. The invention further relates totherapeutic use of the variants of this invention in subjects at riskfor cell damage caused at least in part by apoptosis, and to therapeuticcompositions comprising such mutant proteins, which compositions shouldprovide the desired cytoprotective benefits while carrying a lower riskof bleeding, a side effect of activated protein C therapy.

BACKGROUND OF THE INVENTION

Protein C is a member of the class of vitamin K-dependent serineprotease coagulation factors. Protein C was originally identified forits anticoagulant and profibrinolytic activities. Protein C circulatingin the blood is an inactive zymogen that requires proteolytic activationto regulate blood coagulation through a complex natural feedbackmechanism. Human protein C is primarily made in the liver as a singlepolypeptide of 461 amino acids. This precursor molecule is thenpost-translationally modified by (i) cleavage of a 42 amino acid signalsequence, (ii) proteolytic removal from the one-chain zymogen of thelysine residue at position 155 and the arginine residue at position 156to produce the two-chain form (i.e., light chain of 155 amino acidresidues attached by disulfide linkage to the serine protease-containingheavy chain of 262 amino acid residues), (iii) carboxylation of theglutamic acid residues clustered in the first 42 amino acids of thelight chain resulting in nine gamma-carboxyglutamic acid (Gla) residues,and (iv) glycosylation at four sites (one in the light chain and threein the heavy chain). The heavy chain contains the serine protease triadof Asp257, His211 and Ser360.

Similar to most other zymogens of extracellular proteases and thecoagulation factors, protein C has a core structure of the chymotrypsinfamily, having insertions and an N-terminus extension that enableregulation of the zymogen and the enzyme. Of interest are two domainswith amino acid sequences similar to epidermal growth factor (EGF). Atleast a portion of the nucleotide and amino acid sequences for protein Cfrom human, monkey, mouse, rat, hamster, rabbit, dog, cat, goat, pig,horse, and cow are known, as well as mutations and polymorphisms ofhuman protein C (see GenBank accession P04070). Other variants of humanprotein C are known which affect different biological activities.

Activation of protein C is mediated by thrombin, acting at the sitebetween the arginine residue at position number 15 of the heavy chainand the leucine residue at position 16 (chymotrypsin numbering) (SeeKisiel, J. Clin. Invest., 64:761-769, 1976; Marlar et al., Blood,59:1067-1072, 1982; Fisher et al. Protein Science, 3:588-599, 1994).Other proteins including Factor Xa (Haley et al., J. Biol. Chem.,264:16303-16310, 1989), Russell's viper venom, and trypsin (Esmon etal., J. Biol. Chem., 251:2770-2776, 1976) also have been shown toenzymatically cleave and convert inactive protein C to its activatedform.

Thrombin binds to thrombomodulin, a membrane-bound thrombin receptor onthe luminal surface of endothelial cells, thereby blocking theprocoagulant activity of thrombin via its exosite I, and enhancing itsanticoagulant properties, i.e., activating protein C. As ananticoagulant, activated protein C (APC), aided by its cofactor proteinS, cleaves the activated cofactors factor Va and factor VIIIa, which arerequired in the intrinsic coagulation pathway to sustain thrombinformation (Esmon et al., Biochim. Biophys. Acta., 1477:349-360, 2000a),to yield the inactivated cofactors factor Vi and factor VIIIi.

The thrombin/thrombomodulin complex mediated activation of protein C isfacilitated when protein C binds to the endothelial protein C receptor(EPCR), which localizes protein C to the endothelial cell membranesurface. When complexed with EPCR, APC's anticoagulant activity isinhibited; APC expresses its anticoagulant activity when it dissociatesfrom EPCR, especially when bound to negatively charged phospholipids onactivated platelet or endothelial cell membranes.

Components of the protein C pathway contribute not only to anticoagulantactivity, but also to anti-inflammatory functions (Griffin et al., Sem.Hematology, 39:197-205, 2002). The anti-inflammatory effects ofthrombomodulin, recently attributed to its lectin-like domain, canprotect mice against neutrophil-mediated tissue damage (Conway et al.,J. Exp. Med. 196:565-577, 2002). The murine centrosomal protein CCD41 orcentrocyclin, involved in cell-cycle regulation is identical to murineEPCR lacking the first N-terminal 31 amino acids (Rothbarth et al., FEBSLeft., 458:77-80, 1999; Fukodome and Esmon, J. Biol. Chem.,270:5571-5577,1995). EPCR is structurally homologous to the MHC class1/CD1 family of proteins, most of which are involved in inflammatoryprocesses. This homology suggests that the function of EPCR may not belimited to its ability to localize APC or protein C on the endothelialmembrane (Oganesyan et al., J. Biol. Chem., 277:24851-24854, 2002). APCprovides EPCR-dependent protection against the lethal effects of E. coliinfusion in baboons (Taylor et al., Blood, 95:1680-1686, 2000) and candownregulate proinflammatory cytokine production and favorably altertissue factor expression or blood pressure in various models (Shu etal., FEBS Lett. 477:208-212, 2000; Isobe et al., Circulation,104:1171-1175, 2001; Esmon, Ann. Med., 34:598-605, 2002).

Inflammation is the body's reaction to injury and infection. Three majorevents are involved in inflammation: (1) increased blood supply to theinjured or infected area; (2) increased capillary permeability enabledby retraction of endothelial cells: and (3) migration of leukocytes outof the capillaries and into the surrounding tissue (hereinafter referredto as cellular infiltration) (Roitt et al., Immunology, Grower MedicalPublishing, New York, 1989).

Many serious clinical conditions involve underlying inflammatoryprocesses in humans. For example, multiple sclerosis (MS) is aninflammatory disease of the central nervous system. In MS, circulatingleukocytes infiltrate inflamed brain endothelium and damage myelin, withresultant impaired nerve conduction and paralysis (Yednock et al.,Nature 366:63-66 (1992)). Systemic lupus erythematosus (SLE) is anautoimmune disease characterized by the presence of tissue damage causedby self antigen directed antibodies. Auto-antibodies bound to antigensin various organs lead to complement-mediated and inflammatory cellmediated tissue damage (Theofilopoulos, A. N., Encyclopedia ofImmunology, pp. 1414-1417 (1992)).

APC has not only anticoagulant and anti-inflammatory activities but alsoanti-apoptotic activity. EPCR has been found to be a required cofactorfor the anti-apoptotic activity of APC in certain cells, as APCactivation of protease activated receptor-1 (PAR-1) is EPCR-dependent(Riewald et al., Science, 2296:1880-1882, 2002; Cheng et al., Nat. Med.,9:338-342, 2003; Mosnier and Griffin, Biochem. J., 373:65-70, 2003). APCalso has been shown potentially to inhibit staurosporine-inducedapoptosis in endothelial cells in vitro by modulating the expression ofNFκB subunits (Joyce et al., J. Biol. Chem., 276:11199-11203, 2001).Staurosporine-induced apoptosis in human umbilical vein endothelialcells (HUVEC) and tumor necrosis factor-α-mediated injury of HUVEC,based on transcriptional profiling, suggest that APC's inhibition ofNFκB signaling causes down regulation of adhesion molecules (Joyce etal., supra, 2001). APC's induction of anti-apoptotic genes (e.g.,Bcl2-related protein A1 or Bcl2A1, inhibitor of apoptosis 1 or clAP1,endothelial nitric oxide synthase or eNOS) has been interpreted as apossible mechanism linked to APC's anti-apoptotic effects in astaurosporine model of apoptosis.

APC has a remarkable ability to reduce all-cause 28-day mortality by 19%in patients with severe sepsis (Bernard et al., New Engl. J. Med.344:699-709, 2001a), whereas, potent anticoagulant agents such asantithrombin III and recombinant TFPI have failed in similar phase IIIclinical trials (Warren et al., JAMA, 286:1869-1878, 2001; Abraham etal., Crit. Care Med., 29:2081-2089). The explanation for this differencemay lie in the recently described anti-apoptotic activity of APC, aswell as its anti-inflammatory activity. The clinical success of APC intreating sepsis may be related to its direct cellular effects thatmediate its anti-apoptotic or anti-inflammatory activity.

In spite of the numerous in vivo studies documenting the beneficialeffects of APC, there is limited information about the molecularmechanisms responsible for APC's direct anti-inflammatory andanti-apoptotic effects on cells. APC can directly modulate geneexpression in human umbilical vein endothelial cells (HUVEC) withnotable effects on anti-inflammatory and cell survival genes (Joyce etal., supra, 2001; Riewald et al., supra, 2002). Riewald et al. haveshown this direct effect of APC on certain cells requires PAR-1 and EPCR(Riewald et al., supra, 2002), although they provided no data thatrelated APC functional activity with PAR-1-signaling.

Recombinant activated protein C (rAPC), similar to Xigris (Eli Lilly &Co.), is approved for treating severe sepsis and it may eventually haveother beneficial applications. However, clinical studies have shown APCtreatment to be associated with increased risk of serious bleeding. Thisincreased risk of bleeding presents a major limitation of APC therapy.If APC's effects in sepsis can be attributed to its anti-inflammatoryand cell survival activities, a compound that retains the beneficialanti-inflammatory or cytoprotective activity but has a lessanticoagulant activity is desirable.

SUMMARY OF THE INVENTION

It is an object of this invention to provide variants (mutants) ofrecombinant APC and prodrugs (e.g., variants of recombinant protein C)as therapeutics or research tools for use in alleviating or preventingcell damage associated at least in part with apoptosis. It is also anobject of this invention to provide a method of alleviating orpreventing cell damage associated at least in part with apoptosis,especially in subjects at risk for or suffering from such cell damage.Another object of this invention is to provide a means for screeningcandidate mutants for use in accordance with the invention.

The invention is directed to variants of recombinant APC and prodrugs(protein C variants) that provide reduced anticoagulant activityrelative to anti-apoptotic activity compared to wild-type, and,therefore, have use as cytoprotective agents. Two examples of suchrecombinant APC mutants are KKK191-193AAA-APC (mutation of lysines 191,192 and 193 to alanines) and RR229/230AA-APC (mutation of arginines 229and 230 to alanines). As we demonstrate herein, these exemplary APCvariants retain the desirable property of normal anti-apoptotic,cytoprotective activity but provide significantly reduced risk ofbleeding, given their reduced anticoagulant activity. The APC andprotein C variants of the invention provide a ratio of anti-apoptotic toanticoagulant activity greater than that of wild-type APC (i.e., >1.0).

In one embodiment of the invention, a method of preventing oralleviating damage associated at least in part with apoptosis isprovided. In a related aspect of this embodiment, a method of treatingsubjects at risk for cell damage associated at least in part withapoptosis is provided. These subjects include patients at risk of damageto blood vessels or tissue in various organs caused, at least in part,by apoptosis. At risk patients include, for example, those suffering(severe) sepsis, ischemia/reperfusion injury, ischemic stroke, acutemyocardial infarction, acute or chronic neurodegenerative diseases, orthose undergoing organ transplantation or chemotherapy, among otherconditions. The APC variants and prodrugs of the invention should beuseful in treating subjects who will benefit from APC protectiveactivities that are independent of APC's anticoagulant activity. Prodrugembodiments of this invention may involve recombinant protein C variantsthat, following conversion of protein C to APC, exhibit reducedanticoagulant activity while retaining normal or near-normal cellprotective activities. For example, variants of protein C, whenactivated, will have the desired ratio of anti-apoptotic toanticoagulant activity of greater than 1.0.

In another embodiment of the invention, the APC mutants may be providedas therapeutics or in therapeutic compositions, to offer beneficialcytoprotective effects in cells, while carrying much less risk ofbleeding. In yet another embodiment of the invention, methods ofscreening candidate recombinant APC variants having reducedanticoagulant activity, but retaining the beneficial cell protective andanti-inflammatory activities are provided.

Given the risk of bleeding associated with wild type activated proteinC, the APC mutants of this invention offer advantages over currentlyavailable wild-type recombinant APC. Therefore, APC mutants of theinvention are expected to provide superior therapy, either alone oradjunctive to other agents, whenever APC might be used for itsanti-inflammatory or anti-apoptotic (cell survival) activities, ratherthan purely for its anticoagulant activity.

DESCRIPTION OF DRAWINGS

FIGS. 1 a-1 b: Inhibition of staurosporine-induced (STS) apoptosis inEahy926 endothelial cells by wild-type (rwt-APC) and variants ofrecombinant APC. FIG. 1 a: dose-dependent reduction in STS-inducedapoptosis expressed as percent apoptotic cells. FIG. 1 b: dose-dependentreduction in STS-induced apoptosis with data normalized as percentapoptotic cells relative to control STS (no APC).

FIG. 2: Ratio of anti-apoptotic (cytoprotective) activity toanticoagulant activity for wild-type and variants of recombinant APC.

FIG. 3 a-3 b: Amidolytic and anticoagulant activity of rwt-APC and APCvariants. a, Amidolytic activity of rwt-APC and APC variants against thesmall chromogenic substrate, S-2366. b, Anticoagulant activity ofrwt-APC and APC variants determined using Activated PartialThromboplastin Time (APTT) assays. Each point represents the mean±S.E.M.from at least three independent experiments. Symbols denote: □, rwt-APC;∘, RR229/230AA-APC; ⋄, KKK191-193AAA-APC; ▪, S360A-APC.

FIG. 4 a-4 c: Anti-apoptotic activity of rwt-APC and anticoagulantlyimpaired APC variants. a, Inhibition of staurosporine (STS)-inducedapoptosis by APC (see Methods). Percentage of apoptotic endothelialcells observed in the absence of added APC (18% of all cells) was takenas 100%. Each point represents the mean±S.E.M. from at least threeindependent experiments. Symbols used denote: □, rwt-APC; ∘,RR229/230AA-APC; ⋄, KKK191-193AAA-APC; ▪, S360A-APC; , nostaurosporine. b, c, Reduction of activated caspase-3-positive cells byrwt-APC and APC variants (25 nM, 5 h) upon induction of apoptosis bystaurosporine (2 μM, 4 h). b, Activated caspase-3-positive cellsexpressed as a percentage of the total number of cells present. Asindicated by the “no STS”, thin line, approximately 2% of theendothelial cells were positive for activated caspase-3 in the absenceof staurosporine. Each bar represents the mean±SEM of two to fourindependent experiments. c, Immunofluorescence analysis of activatedcaspase-3-positive cells using an activated caspase-3 specific antibody(red) and DAPI nuclear staining (blue). Columns represent identicalfields. Original magnification was 200×.

FIG. 5: Inhibition of apoptosis by rwt-APC and APC variants requiresPAR-1 and EPCR. PAR-1 and EPCR-dependence for inhibition ofstaurosporine-induced endothelial cell apoptosis by rwt-APC andanticoagulantly impaired APC variants was studied using blockingantibodies against PAR-1 (open bars) (combination of WEDE-15 at 20 μg/mland ATAP-2 at 15 μg/ml) or EPCR (cross-hatched bars) (rabbit anti-EPCRat 20 μg/ml). Solid bars represent “no antibodies added”. Cells wereincubated with rwt-APC or APC variants (5 nM) 5 h prior to induction ofapoptosis by staurosporine (10 μM, 1 h). Apoptosis was analysed by theuptake of Apopercentage dye and expressed as a percentage relative tothe percentage of apoptotic cells observed in the absence of added APC(20% of all cells), which was set as 100%. The bar with “vertical lines”represents relative apoptosis in the absence of APC and staurosporine.Each bar represents the mean±S.E.M. from at least three independentexperiments.

FIG. 6: Cleavage of PAR-1 N-terminal TR33-62 peptide at Arg41 by rwt-APCand APC variants. HPLC was used to monitor TR33-62 cleavage by APC overtime as disappearance of the TR33-62 peptide substrate peak (opensymbols) and as appearance of the TR42-62 peptide product peak (solidsymbols). Symbols denote: ▪,□: rwt-APC; ,∘: RR229/230AA-APC; ♦,⋄:KKK191-193AAA-APC and X,X: S360A-APC. The pooled data points of 3-5independent experiments are shown for rwt-APC and the two anti-apoptoticAPC variants. No cleavage was observed for the S360A-APC that lacks theactive site Ser (X). Error bars indicate ±S.E.M.

DETAILED DESCRIPTION OF THE INVENTION

Activated protein C (APC) has traditionally been regarded as ananticoagulant enzyme in the coagulation cascade, inhibiting thrombinformation and subsequent fibrin-clot formation by inactivating thecofactors factor Va and factor VIIIa (Esmon, supra, 2000a). However, APCalso has the remarkable ability to reduce mortality in severe sepsis(Bernard et al., supra, 2001a; Bernard et al., Crit. Care Med.,29:2051-59, 2001b; Hinds, Brit. Med. J., 323:881-82, 2001; Kanji et al.,Pharmacother., 21:1389-1402, 2001), while other anticoagulants such asantithrombin III and tissue factor pathway inhibitor have failed in thiscapacity (Warren et al., supra, 2001; Abraham et al., supra, 2001). Thisproperty of APC has peaked investigators' interest in the lessextensively studied direct anti-inflammatory and anti-apoptoticactivities attributed to APC (see, e.g., Cheng et al. Nat. Med.,9:33842, 2003; Domotor et al., Blood, 101:47974801, 2003; Fernandez etal., Blood Cells Mol. Dis., 30:271-276, 2003; Esmon, J. Autoimmun.,15:113-116, 2000b). APC also has potential to protect the brain fromdamage caused by ischemic stroke (Cheng et al., supra, 2003; EsmonThrombos Haemostas, 83:639-643, 2000c).

A major concern for the use of APC as a therapeutic is an increased riskof bleeding complications (Bernard et al., supra, 2001a; Bernard et al.,supra, 2001b) due to APC anticoagulant activity. The APC variants ofthis invention solve this problem by having reduced anticoagulantactivity over endogenous APC or wild-type recombinant APC, whileretaining beneficial anti-apoptotic activity. Differentiating theanticoagulant activity from the anti-apoptotic activity was the firststep in solving this problem. We have focused in part on the role ofEPCR in regulation of these activities.

EPCR was originally discovered as a receptor capable of binding proteinC and APC with equal affinities (Fukodome and Esmon, supra, 1995), andEPCR was shown to enhance the activation of protein C by thethrombin-thrombomodulin complex (Stearns-Kurosawa, et al., Proc. Nat'lAcad. Sci., USA, 93:10212-10216, 1996), apparently by optimizing thespatial localization of protein C for efficient activation bythrombomodulin-bound thrombin. Presumably EPCR binds APC to theendothelial surface and positions APC's active site proximate to thePAR-1 cleavage site at Arg41. Paradoxically, although EPCR functionmight be anticoagulant by stimulating protein C activation(Stearns-Kurosawa, et al., supra, 1996), APC anticoagulant activity isactually inhibited when APC is bound to EPCR (Regan et al., J. Biol.Chem., 271:17499-17503, 1996). Because binding of APC to EPCR isessential for APC's anti-apoptotic activity, we have concluded that theanti-apoptotic activity of APC is independent of its anticoagulantactivity. We hypothesized that certain APC mutants could be generatedwhich lack anticoagulant activity but retain anti-apoptotic activity.Such mutants could be therapeutically useful if they provided patientswith direct cell survival activity without increased risks of bleeding.

We have determined the structural elements of APC required for itsanti-apoptotic activity, by assaying different forms of APC for theiranti-apoptotic activity. The staurosporine-induced apoptosis was blockedby pretreatment of APC with an anti-APC monoclonal antibody or heatdenaturation of APC, thereby establishing the specificity of APC'santi-apoptotic activity (Mosnier and Griffin, supra, 2003). APC-mediatedinhibition of staurosporine-induced apoptosis was found to require APC'sactive site, since the inactive protein C zymogen, as well as aninactive APC mutant, in which the active site Ser was replaced by Ala,S360A-APC (Gale et al., Protein Sci., 6:132-140, 1997), were devoid ofanti-apoptotic activity (Mosnier and Griffin, supra, 2003). This impliesthat the anti-apoptotic activity of APC is mediated by proteolysis.

It was not known whether the APC-mediated inhibition ofstaurosporine-induced apoptosis (Joyce et al., supra, 2001) wasdependent on PAR-1 and EPCR, until we demonstrated that inhibition ofstaurosporine-induced apoptosis by APC was dependent on PAR-1 and EPCRusing a modified staurosporine-induced apoptosis model with EAhy926endothelial cells (Mosnier and Griffin, supra, 2003). Inhibition ofhypoxia-induced apoptosis in human brain endothelial cells also has beenshown to require PAR-1 (Cheng et al., supra, 2003). Thus, consistentwith the implication that APC's proteolytic active site is required forinhibition of apoptosis, preincubation of cells with blocking antibodiesagainst PAR-1, but not against PAR-2, abolished APC-mediated inhibitionof staurosporine-induced apoptosis (Mosnier and Griffin, supra, 2003).Furthermore, APC anti-apoptotic activity was abolished by an anti-EPCRantibody that blocks binding of APC to EPCR (Mosnier and Griffin, supra,2003), and controls showed that this effect of the anti-EPCR antibodywas neutralized by preincubation of the antibody with its peptideimmunogen (Mosnier and Griffin, supra, 2003). Therefore, based onantibody blocking studies, PAR-1 and EPCR are required for APC toinhibit staurosporine-induced apoptosis of endothelial cells.

This requirement for PAR-1 and EPCR for inhibition ofstaurosporine-induced apoptosis of EAhy926 endothelial cells also isconsistent with the finding that these receptors are important for APC'santi-apoptotic activity in the setting of hypoxic brain microvascularendothelial cells (Cheng et al., supra, 2003).

APC can cleave a synthetic extracellular N-terminal PAR-1 polypeptide atArg41, the thrombin cleavage site (Kuliopulos et al., Biochemistry,38:4572-4585, 1999). Cleavage of this synthetic PAR-1 polypeptide by APCis 5,000-times slower than by thrombin (Kuliopulos et al., supra, 1999).When thrombin cleaves PAR-1 at Arg41, potent cell signaling pathwaysmight be initiated. It is likely that APC cleavage of PAR-1 at Arg41initiates cell signals, including phosphorylation of MAP kinase (Riewaldet al., supra, 2002). In brain endothelial cells subjected to hypoxia,an early result of APC signaling is the inhibition of increases in thelevels of p53 (Cheng et al., supra, 2003). Previous studies suggest thatAPC directly alters the gene expression profiles of HUVEC so thatseveral anti-apoptotic genes are upregulated (Joyce et al., supra, 2001;Riewald et al., supra, 2002) and that APC specifically downregulateslevels of the pro-apoptotic factor, Bax, while it upregulates levels ofthe anti-apoptotic factor, Bcl-2, in brain endothelial cells (Cheng etal., supra, 2003). The specific alteration of the critical ratio ofBax/Bcl-2 is likely of key importance for apoptosis. Other than theseevents, little can be stated about the mechanisms for PAR-1-dependentAPC signaling. It is interesting to note that the PAR-1 agonist peptide,TFLLRNPNDK, exhibited no protection from staurosporine-induced apoptosisof EAhy926 cells whereas this agonist provided partial rescue of brainendothelial cells from hypoxia-induced apoptosis, suggesting there aresubtle, but significant, differences between APC's PAR-1-dependentanti-apoptotic activities in these two models.

In vivo data are consistent with an important distinction between theanticoagulant and cell protective activities of APC. APC-inducedneuroprotective effects in a murine ischemia/reperfusion injury modelwere observed at low APC doses that had no effect on fibrin depositionor on restoration of blood flow, indicating that APC's neuroprotectiveeffects, at least in part, were independent of APC's anticoagulantactivity (Cheng et al., supra, 2003).

No inhibition of staurosporine-induced apoptosis of EAhy926 cells wasobserved with either PAR-1 or PAR-2 agonist peptides in the absence ofAPC. Moreover, thrombin, the archetype activator of PAR-1, did notinhibit staurosporine-induced apoptosis (Mosnier and Griffin, supra,2003). The failure of these other activators of PAR-1 to provide cellsurvival activity indicates that the PAR-1-dependent anti-apoptoticeffects of APC for staurosporine-induced apoptosis are specific for APC.Without being bound to a mechanism of action, we can speculate that whenEPCR-bound APC cleaves and activates PAR-1, a significant modulation ofPAR-1's intracellular signaling occurs, compared to signals triggered bythrombin or the PAR-1 agonist peptide. Another potential source ofcomplexity may arise from the reported ability of EPCR to mediatenuclear translocation of APC (Esmon, supra, 2000c). The intracellularsignals and pathways that cause inhibition of apoptosis by APC invarious cell model systems remain to be elucidated.

The physiological relevance of APC EPCR-dependent signaling via PAR-1 isfurther demonstrated by the APC-induced neuroprotective effects in amurine ischemia/reperfusion injury model that requires PAR-1 and EPCR(Cheng et al., supra, 2003). APC may act via the EPCR and PAR-1 onstressed brain endothelial cells, or the PAR-1 and the proteaseactivated receptor-3 (PAR-3) on stressed neurons, to activateanti-apoptotic pathways and/or pro-survival pathways in these stressedand/or injured brain cells. In human brain endothelium in vitro and inanimals in vivo (ischemic stroke and NMDA models), APC can inhibit thep53-signaling pro-apoptotic pathway in stressed or injured brain cells(International Patent Application No. PCT/US03/38764).

Examples

Structure-activity relationships of protein C and activated protein Cmay be studied using variant polypeptides produced with an expressionconstruct transfected in a host cell with or without expressingendogenous protein C. Thus, mutations in discrete domains of protein Cor activated protein C may be associated with decreasing or evenincreasing activity in the protein's function.

To generate the APC variants and prodrugs of this invention, whichprovide a reduced risk of bleeding, i.e., reduced anticoagulantactivity, but that retain useful cytoprotective activities, we havedissected anticoagulant activity from anti-apoptotic activity of APC bysite-directed mutagenesis. Several amino acids in various surface loopsof the protease domain of APC were identified that, when mutated toalanine, severely reduced anticoagulant activity but did not affectanti-apoptotic activity. These unexpected findings indicate thatstrategies aimed at reducing the anticoagulant activity while preservingthe anti-apoptotic activity of APC are feasible and worthwhile, becausethey are likely to reduce bleeding complications associated with thecurrent and future clinical uses of recombinant APC variants whichretain cytoprotective activities.

The structural basis of APC's anticoagulant activity has been centeredprimarily on the interaction of APC with factor Va. APC cleavage siteswithin factor Va are located at residues Arg³⁰⁶, Arg⁵⁰⁶ and Arg⁶⁷⁹ andcleavage of the former two correlates with loss of cofactor activity(Rosing and Tans, Thromb Haemost, 78:427-433, 1997; Kalafatis and Mann,J. Biol. Chem., 268:27246-57, 1993). Cleavage of factor Va at Arg506occurs rapidly and usually precedes cleavage at Arg³⁰⁶. It is thereforeconsidered the predominant site for the initial inactivation of thefactor Va molecule (Norstrom et al., J. Biol. Chem., 278:24904-1133,2003; Nicolaes, et al., J. Biol. Chem., 270:21158-66,1995). Theinteraction of APC with the Arg⁵⁰⁶ cleavage site in factor Va has beenextensively characterized and as a result a factor Va binding site onthe positively charged surface of the protease domain of APC has beendefined (Gale et al., Blood, 96:585-593, 2000; Gale et al., J. Biol.Chem. 277:28836-28840, 2002; Friedrich et al., J. Biol. Chem.,276:23105-08, 2001 a; Knobe et al., Proteins, 35:L218-234, 1999; Shen etal., Thromb. Haemost., 82:72-79, 1999). This positive exosite for factorVa binding on APC is generally located in the same area as the anionbinding exosite I of thrombin and is comprised of residues in loop 37,which contains protein C residues 190-193 (equivalent to chymotrypsin(CHT) residues 36-39), the calcium ion-binding loop containing residues225-235 (CHT 70-80) and the autolysis loop containing residues 301-316(CHT 142-153) (Mather et al., EMBO J., 15:6822-31, 1996). In addition,mutations in loop 60, containing protein C residues 214-222 (CHT 60-68)have little effect on factor Va inactivation by APC although this loopis implicated in interactions with thrombomodulin and heparin (Gale etal., supra, 2002; Friedrich et al., supra, 2001 a; Knobe et al., supra,1999; Shen et al., supra, 1999; Friedrich et al., J. Biol. Chem.,276:24122-28, 2001b).

Gale et al. (supra, 2002) have demonstrated that mutations in thesurface loops of APC affect its anticoagulant activity. APC mutantsKKK191-193AAA (loop 37), RR229/230AA (calcium loop), RR306/312AA(autolysis loop), RKRR306-314AAAA (autolysis loop) were shown to have10%, 5%, 17%, and less than 2% of the anticoagulant activity of nativeAPC, respectively. Subsequently, we found that these APC mutants withreduced anticoagulant activity (i.e., KKK191-193AAA, RR229/230AA(Mosnier et al. (Blood epup, 2004)) and RR306/312AA (Mosnier & Griffin,unpublished observations)) retain the anti-apoptotic activity of APC instaurosporine model of apoptosis.

To demonstrate that we could distinguish between structural features ofAPC necessary for anticoagulant activity versus cell-protectiveactivity, we studied recombinant variant forms of APC that had severelyreduced anticoagulant activity. Using double, triple and quadruplecombinations of site-directed mutations in the factor Va binding site ofAPC, we constructed a set of APC variants with severely decreasedanticoagulant activity but with essentially unchanged enzymatic activityfor small peptide (chromogenic) substrates (Gale et al., supra, 2000;Gale et al., supra, 2002). Anticoagulant activity was determined in adilute prothrombin clotting assay (Gale et al., supra, 2002). Thecytoprotective (anti-apoptotic) activity of APC mutants was tested in astaurosporine-induced model of apoptosis with EAhy926 endothelial cells,with the modifications described by Mosnier and Griffin (supra, 2003).It was discovered that APC-mediated inhibition of staurosporine-inducedapoptosis required APC's active site, since the inactive APC mutant inwhich the active site serine360 was replaced by alanine (S360A-APC, seeTable 1) (Gale et al., supra, 1997), was devoid of anti-apoptoticactivity (Mosnier and Griffin, supra, 2003) (FIGS. 1 a and 1 b).Recombinant APC inhibition of staurosporine-induced apoptosis in Eahy926endothelial cells was determined by Apopercentage staining. Inhibitionof apoptosis by recombinant wild-type APC (rwt-APC) was dose-dependent(FIG. 1 a). Half-maximum inhibition of staurosporine-induced apoptosiswas achieved at 0.24 nM rwt-APC, using a 5 hour preincubation of APCwith cells before addition of staurosporine. Note the absence ofapoptotic activity in the S360A mutant (FIG. 1 b). The mutationsdescribed in examples 1-3 and their % activities relative to wild-typeare indicated in Table 1. Also indicated in Table 1 is the ratio ofanti-apoptotic (cytoprotective) activity to anticoagulant activity foreach of wild-type APC and mutant APC of examples 1-3, as described inexample 4.

TABLE 1 Overview of APC mutants wt-APC sequence cytoprotectiveanticoagulant Cytoprotective- FVa inact. Amydolytic (underlined areactivity activity anticoagulant Arg⁵⁰⁶ (Arg³⁰⁶) activity¹ T½ mutantmutated to alanine) (% wt-APC) (% wt-APC)¹ ratio (% wt-APC)¹ (% wt-APC)(min) rwt-APC n/a 100% 100%  1.0 100% (100%) 100% 21.4 229/230-APC225-EYDLRRWEKWE-235  89% 6.6% 13.5 25% (110%) 115% 27.6 3K3A-APC189-DSKKKL-194 120%  15% 8.0 11% (67%) 134% 20.7 306-314-APC305-SREKEAKRNRT-315  <1%  1.6%² 0.6 1.4% (16%) 75.6%  46.2(anticoagulant activity determined by dilute prothrombin time (PT))¹from references (Gale et al., 1997; Gale et al., 2000; Gale et al.,2002); See text and Methods for more information. ²determined by APTTinstead of dilute PT n/a: not applicable; rwt-APC, recombinantwild-type-APC; 229/230-APC, RR229/230AA-APC; 3K3A-APC,KKK191-193AAA-APC; 306-314-APC, RKRR306/311/312/314AAAA-APC.

Example 1

Replacing the two arginine residues, Arg229 and Arg230, in thecalcium-binding loop of APC with alanine residues resulted in a form ofAPC RR229/230AA-APC (229/230-APC), see Table 1) with only 6.6% residualanticoagulant activity. This reduction in anticoagulant activity ofRR229/230AA-APC was primarily due to reduced inactivation of factor Va(FVa) at Arg⁵⁰⁶ whereas cleavage of factor Va at Arg³⁰⁶ was much lessaffected.

The dose-dependence for inhibition of apoptosis by RR229/230AA-APC(FIGS. 1 a and 1 b) was similar to that of recombinant wild type(rwt)-APC. Half-maximum inhibition of staurosporine-induced apoptosis byRR229/230AA-APC was achieved at 0.27 nM. This example demonstrates thatthe anticoagulant activity of APC is not required for the cytoprotective(anti-apoptotic) activity of APC.

Example 2

In this example, an APC mutant in which three consecutive lysineresidues in loop 37 were replaced with three alanines KKK191-193AAA-APC(3K3A-APC), see Table 1) displayed only 15% residual anticoagulantactivity as determined in a dilute prothrombin clotting assay (Gale etal., supra, 2002). The reduction in anticoagulant activity ofKKK191-193AAA-APC was due to severely reduced cleavage of factor Va atArg⁵⁰⁶ (11% of rwt-APC), whereas inactivation of factor Va at Arg³⁰⁶ wasonly moderately affected (67% of rwt-APC) (Table 1). Remarkably, as seenin FIGS. 1 a and 1 b, the anti-apoptotic activity of KKK191-193AAA-APCwas similar to that of rwt-APC with half-maximum inhibition ofstaurosporine-induced apoptosis at 0.20 nM.

Example 3

In this example, four out of the five basic amino acids in the so-calledautolysis loop of APC were replaced by alanine residues, resulting in aform of APC RKRR306/311/312/314AAAA-APC (306-314-APC, see Table 1)having only 1.6% residual anticoagulant activity, as determined by theactivated partial thromboplastin time (APTT) [765]. The reduction inanticoagulant activity of RKRR306/311/312/314AAAA-APC was due toseverely reduced cleavage of factor Va at Arg⁵⁰⁶ (1.4% of rwt-APC)whereas inactivation of factor Va at Arg³⁰⁶ was only moderately affected(16% of rwt-APC). The RKRR306/311/312/314AAAA-APC mutant was severelydeficient in cytoprotective (anti-apoptotic) activity (FIGS. 1 a and 1b), with inhibition of staurosporine-induced apoptosis requiring muchhigher concentrations of this mutant APC compared to rwt-APC or theother two APC mutants, RR-229/230AA-APC and KKK191-193AAA-APC.

Example 4

The ratio of anti-apoptotic activity to anticoagulant activity wascalculated for rwt-APC and for each APC mutant of examples 1-3 (seeTable 1), based on the anti-apoptotic activity data in FIG. 1 andpublished anticoagulant activities (Gale et al., supra, 2000; Gale etal., supra, 2002). The ratio of activities for rwt-APC is defined as1.0. These ratios, as shown in FIG. 2, indicate that APC mutants withmutations in certain residues in certain protease domain surface loopscan exhibit 8-times to 14-times greater anti-apoptotic activity relativeto anticoagulant activity. The two mutants, KKK191-193AAA-APC andRR229/230AA-APC, would be expected to exhibit anti-apoptotic orcytoprotective activity comparable to rwt-APC while having an 8-fold to14-fold reduced risk of bleeding because of the reduction inanticoagulant activity.

The ratio of anti-apoptotic to anticoagulant activity of a recombinantAPC mutant may be used to identify variants of recombinant APC of thisinvention having therapeutic potential. A ratio of >1.0 is indicative ofa therapeutic recombinant APC mutant having cytoprotective benefits andreduced risks of bleeding for a subject in need of acute or prophylactictreatment for cell damage, in accordance with this invention.Preferably, a therapeutic variant of recombinant APC would have a ratioof anti-apoptotic activity to anticoagulant activity of greater thanabout 2. More preferably, said ratio would be greater than about 4. Mostpreferably, said ratio would be greater than about 8.

Prodrug embodiments of this invention may involve recombinant protein Cvariants that, following conversion of protein C to APC either in vitroor in vivo, exhibit reduced anticoagulant activity while retainingnormal or near-normal cell protective activities, i.e., have a ratio ofanti-apoptotic:anticoagulant activity greater than 1.0. Preferably, theprodrugs of the invention may be converted to APC variants that have aratio of anti-apoptotic activity to anticoagulant activity that isgreater than about 2, more preferably the ratio is greater than about 4or most preferably the ratio is greater than about 8.

The invention comprises several embodiments which are described below.

In one embodiment, the variants of APC of the invention may be used ineffective doses to provide cytoprotection to cells at risk forundergoing apoptotic cell death or stress-induced injury either in vivoor in vitro. In an aspect of this embodiment the APC variants may beadministered in therapeutic doses to subjects who could benefit fromAPC's cytoprotective activities that are independent of theanticoagulant activity. Such subjects comprise patients at risk fordamage to blood vessels or other tissue organs, which damage is causedat least in part by apoptosis. The risk for cell damage may be theresult of any one or more of sepsis, ischemia/reperfusion injury,stroke, ischemic stroke, acute myocardial infarction, acuteneurodegenerative disease, chronic neurodegnerative disease, organtransplantation, chemotherapy, or brain radiation injury. These causesof cell damage are not intended in any way to limit the scope of theinvention, as one skilled in the art would understand that otherdiseases or injuries also may put cells at risk for damage caused atleast in part by apoptosis. The effective doses or therapeutic doseswill be those that are found to be effective at preventing oralleviating cell damage caused at least in part by apoptosis. In anotheraspect of this embodiment, the variants of the invention may be appliedto cells or tissue in vitro or in situ in vivo.

In another embodiment, the variants of APC or prodrugs of the inventionmay be used to formulate pharmaceutical compositions with one or more ofthe utilities disclosed herein. The therapeutic compositions may beadministered in vitro to cells in culture, in vivo to cells in the body,or ex vivo to cells outside of a subject, which may then be returned tothe body of the same subject or another. The cells may be removed from,transplanted into, or be present in the subject (e.g., geneticmodification of endothelial cells in vitro and then returning thosecells to brain endothelium). The prodrugs would be expected to becapable of being converted to APC in situ. Candidate agents may also bescreened in vitro or in vivo to select those with desirable properties.The cell may be from the endothelium (e.g., an endothelial or smoothmuscle cell or from the endothelium of a brain vessel).

Therapeutic compositions comprising the variant APC of the invention maybe provided in dosage form. In one aspect of this embodiment, thetherapeutic compositions of the invention may further comprise apharmaceutically acceptable carrier and may still further comprisecomponents useful for delivering the composition to a subject's brain.Such pharmaceutical carriers and delivery components are known in theart. Addition of such carriers and other components to the compositionof the invention is well within the level of skill in this art. Forexample, a permeable material may release its contents to the local areaor a tube may direct the contents of a reservoir to a distant locationof the brain.

The pharmaceutical compositions of the invention may be administered asa formulation, which is adapted for direct application to the centralnervous system, or suitable for passage through the gut or bloodcirculation. Alternatively, pharmaceutical compositions may be added tothe culture medium. In addition to active compound, such compositionsmay contain pharmaceutically-acceptable carriers and other ingredientsknown to facilitate administration and/or enhance uptake. It may beadministered in a single dose or in multiple doses, which areadministered at different times. A unit dose of the composition is anamount of APC mutants provides cytoprotection, inhibits apoptosis orcell death, and/or promotes cell survival but does not provide aclinically significant anticoagulant effect, a therapeutic level of suchactivity, or has at least reduced anticoagulant activity in comparisonto previously described doses of activated protein C. Measurement ofsuch values are within the skill in the art: clinical laboratoriesroutinely determine these values with standard assays and hematologistsclassify them as normal or abnormal depending on the situation. Examplesof how to measure such values are described below.

The pharmaceutical compositions of the invention may be administered byany known route. By way of example, the composition may be administeredby a mucosal, pulmonary, topical, or other localized or systemic route(e.g., enteral and parenteral). In particular, achieving an effectiveamount of activated protein C, prodrug, or functional variant in thecentral nervous system may be desired. This may involve a depotinjection into or surgical implant within the brain. “Parenteral”includes subcutaneous, intradermal, intramuscular, intravenous,intra-arterial, intrathecal, and other injection or infusion techniques,without limitation.

Suitable choices in amounts and timing of doses, formulation, and routesof administration can be made with the goals of achieving a favorableresponse in the subject (i.e., efficacy or therapeutic), and avoidingundue toxicity or other harm thereto (i.e., safety). Administration maybe by bolus or by continuous infusion. Bolus refers to administration ofa drug (e.g., by injection) in a defined quantity (called a bolus) overa period of time. Continuous infusion refers to continuing substantiallyuninterrupted the introduction of a solution into a blood vessel for aspecified period of time. A bolus of the formulation administered onlyonce to a subject is a convenient dosing schedule, although in the caseof achieving an effective concentration of activated protein C in thebrain more frequent administration may be required. Treatment mayinvolve a continuous infusion (e.g., for 3 hr after stroke) or a slowinfusion (e.g., for 24 hr to 72 hr when given within 6 hr of stroke).Alternatively, it may be administered every other day, once a week, oronce a month. Dosage levels of active ingredients in a pharmaceuticalcomposition can also be varied so as to achieve a transient or sustainedconcentration of the compound or derivative thereof in a subject and toresult in the desired therapeutic response.

Thus, “therapeutic” refers to such choices that involve routinemanipulation of conditions to achieve a desired effect (e.g., inhibitionof apoptosis or cell death, promotion of cell survival, cytoprotection,neuroprotection, or combinations thereof). The amount of mutant proteinC or mutant activated protein C administered to subjects may be higherthan doses of recombinant protein C or activated protein C, if necessaryfor maximal cytoprotection, because of the reduced risk of bleeding. Inthis manner, “therapeutic amount” refers to the total amount ofactivated protein C variant or protein C variant that achieves thedesired cytoprotective effect, but with reduced risk for bleeding due toreduced anticoagulant activity (for bolus administration, e.g., 2 mg/kgor less, 1 mg/kg or less, 0.5 mg/kg or less, 0.04 mg/kg or less, 0.03mg/kg or less, 0.02 mg/kg or less, 0.01 mg/kg or less, 0.005 mg/kg orless, depending on the species of the subject or disease to be treated).

The therapeutic amount may be about 0.01 mg/kg/hr to about 1.1 mg/kg/hr,for example, administered by continuous infusion over 4 hour to 96 hour,to as little as about 0.01 mg/kg/hr to about 0.10 mg/kg/hr for about 24hours. Preferably, the therapeutic dose would be administered bycontinuous infusion for about 4 to about 72 hours. More preferably, bycontinuous infusion for about 4 to about 48 hours. More preferably, bycontinuous infusion for about 12 to about 48 hours. More preferably, bycontinuous infusion for about 12 to about 36 hours. More preferably, bycontinuous infusion for about 4 to about 36 hours. More preferably, bycontinuous infusion for about 12 to about 24 hours. Most preferably, bycontinuous infusion for about 24.

The therapeutic amount may be based on titering to a blood level amountof APC of about 0.01 μg/ml to about 1.6 μg/ml, preferably from about0.01 μg/ml to about 0.5 μg/ml. It is also within the skill of the art tostart doses at levels lower than required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. It is likewise within the skill of the artto determine optimal concentrations of variants to achieve the desiredeffects in the in vitro and ex vivo preparations of the invention, e.g.,about 1-100 nM.

In yet another embodiment, a method of screening candidate agents toidentify other variants of recombinant APC having therapeutic potentialin accordance with the invention is provided. One aspect of thisembodiment comprises mutating the recombinant APC or protein C at anysurface loop of the protease domain and determining the variant APC'santicoagulant and cytoprotective activities in assays as described. Themethod of the embodiment further comprises measuring the anticoagulantactivity of said mutated recombinant activated protein C; measuring theanti-apoptotic activity of said mutated recombinant activated protein C;calculating the ratio of anti-apoptotic activity to anticoagulantactivity; identifying the recombinant activated protein C as potentiallytherapeutic if said ratio is greater than 1.0. Preferably, the ratio isgreater than about 2, more preferably the ratio is greater than about 4,most preferably the ratio is greater than about 8. Where the candidateagent is a prodrug, the prodrug may comprise a protein C variant, whichis capable of being converted to an activated protein C variant eitherin vivo or in vitro. To screen the candidate protein C variant fordesirable properties in accordance with the invention, the protein Cvariant would be converted to the activated form (APC) prior tomeasuring activities.

In another aspect of this embodiment, a library of candidate agents isselected which are variants of recombinant APC or protein C having atleast one mutation at a residue in a protease domain of a surface loopselected from the group consisting of loop 37, calcium loop, andautolysis loop. The method comprises converting the protein C variant toactivated protein C variant; determining anti-apoptotic activity of saidcandidate agents in one or more stressed or injured cells by exposingsaid cells to an apoptotic-inducing concentration of staurosporine inthe presence of an amount of a candidate agent; determininganticoagulant activity of said candidate agents in one or more stressedor injured cells exposing cells to same amount of the same candidateagent as in (b), and performing a dilute prothrombin time clottingassay; calculating the ratio of the anti-apoptotic activity determinedin (a) to the anticoagulant activity of (b); and selecting candidateagents having an anti-apoptotic:anticoagulant activity ratio greaterthan 1.0. Preferably, the ratio is greater than about 2, more preferablythe ratio is greater than about 4, most preferably the ratio is greaterthan about 8.

Other permutations of this basic scheme of screening for candidateagents are within the ordinary skill in the art and are encompassed bythe invention. Examples of such permutations non-exclusively includeusing other methods of inducing apoptosis and other tests for measuringapoptotic activity and anticoagulant activity.

Methods Protein C Activation

Recombinant forms of protein C can be produced with a selected chemicalstructure (e.g., native, mutant, or polymorphic). As an illustration, agene encoding human protein C is described in U.S. Pat. No. 4,775,624and can be used to produce recombinant human protein C as described inU.S. Pat. No. 4,981,952. Human protein C can be recombinantly producedin tissue culture and activated as described in U.S. Pat. No. 6,037,322.Natural human protein C can be purified from plasma, activated, andassayed as described in U.S. Pat. No. 5,084,274. The nucleotide andamino acid sequence disclosed in these patents may be used as areference for protein C.

In the above examples of this invention, recombinant wild-type APC(wt-APR), RR229/230AA-APC (229/230-APC), KKK191/192/193AAA-APC(3K3A-APC), RKRR306/311/312/314AAAA-APC (306-314-APC) and S360A-APC wereprepared as described (Gale et al., supra, 1997; Gale et al., supra,2000; Gale et al., supra, 2002). Protein C was activated by thrombin(3281 U/mg, Enzyme Research Labs, South Bend, Ind.). Protein C in HBS(HEPES buffered saline, 50 mM HEPES, 150 mM NaCl) with 2 mM EDTA and0.5% bovine serum albumin (BSA), pH 7.4, at a concentration of 600 μg/mLwas incubated for 2.5 hours with 12 μg/mL thrombin at 37° C, followed bythe addition of 1.1 units of hirudin (Sigma, St Louis, Mo.) per unit ofthrombin to inactivate the thrombin. Controls were done in amidolyticassays, APTT clotting assays and FVa inactivation assays to verify thatthe thrombin and hirudin used had no effect on subsequent assays.

A “mutation” refers to one or more changes in the sequence ofpolynucleotides and polypeptides as compared to native activated proteinC, and has at least one function that is more active or less active, anexisting function that is changed or absent, a novel function that isnot naturally present, or combinations thereof.

Active-Site Titration of APC

All APC mutants were quantitated using an active site titration adaptedfrom Chase and Shaw (Biochem. Biophys. Res. Commun., 29:508-514,1967)using APC at approximately 8 μM in HBS and p-nitrophenol-guanidinobenzoate at 0.1 mM with an extinction coefficient for p-nitrophenol of11400 M⁻¹ cm⁻¹ calculated for pH 7.4.

Kinetic Analysis of APC

Michaelis constants (K_(m)) and catalytic rate constants (k_(cat)) forthe chromogenic substrate, Pefachrome PCa (Pentapharm, Basel,Switzerland), were determined by varying substrate concentration from1.43 mM to 0.0446 mM in HBS, 0.5% BSA, 5 mM CaCl₂, pH 7.4 with APC at5.7 nM. Michaelis constants were derived using Eadie-Hofstee plots.Alternatively, the 5 mM CaCl₂ was replaced with 5 mM EDTA for similardeterminations. Color development was measured with an Optimaxmicroplate reader (Molecular Devices, Sunnyvale, Calif.) (Mesters etal., J. Biol. Chem., 266:24514-19,1991).

Cell Culture

EAhy926 endothelial cells were obtained from Dr. C. J. S. Edgell(University of North Carolina, Chapel Hill N.C.) and were maintained inDMEM high glucose (Gibco, Grand Island, N.Y.) with 10% fetal bovineserum (Omega Scientific, Tarzana, Calif.), 100 U/ml penicillin G sodium(Gibco), 100 μg/ml streptomycin sulphate (Gibco) and 2 mM glutamine(Gibco) at 37° C. in a humid atmosphere containing 5% CO₂ in air asdescribed (Edgell et al., Proc. Nat'l Acad. Sci., USA, 80:3734-3737,1983).

Apoptosis Assay

Staurosporine-induced apoptosis of endothelial cells was initiated usingour modifications (Mosnier and Griffin, supra, 2003) of the previouslydescribed assay (Joyce et al., supra, 2001). The modifications involvedculturing the cells on gelatin-coated coverslips, changing thestaurosporine concentration and optimizing the APC preincubation timesbefore addition of staurosporine as described below. Staurosporine, anATP analogue and inhibitor of protein kinase C (PKC), is a well knownand potent inducer of apoptosis. Apopercentage dye is a measurement ofexpression of phosphatidylserine on the outside surface of the cellmembrane and is therefore similar to what is measured by traditionalannexin-V labeling. The transfer of phosphatidylserine to the outsidesurface of the cell membrane permits the unidirectional transport of theApopercentage dye inside the cell where it is retained and accumulates.The accumulated dye has a red/purple color and is visible under aconventional microscope (Joyce et al., supra, 2001; Mosnier and Griffin,supra, 2003). We used this dye to monitor apoptosis. Alternatively,cells may be incubated with the apoptosis specific dye, YO-PRO-1 (10 μM,5 min) (Molecular Probes, Eugene, Oreg.) as described (Idziorek et al.,J. Immunol. Methods, 185:249-258, 1995) or for 20 min with the syntheticsubstrate for caspase 3-like enzymes, DEVD-amc (Calbiochem, San Diego,Calif.). Staurosporine induced a time-dependent andconcentration-dependent apoptosis in EAhy926 endothelial cells, asdetermined by Apopercentage staining (data not shown).

Briefly, 12 mm round coverslips (Fisherbrand, Pittsburgh, Pa.) were acidwashed, rinsed with distilled water and 95% ethanol, dipped 10× ingelatin (0.5% gelatin provided with the Apopercentage dye) until anhomogenous drop was formed and air dried. EAhy926 cells were grown toconfluency on gelatin-coated coverslips in 24 well plates and incubatedwith APC for 5 hours prior to apoptosis induction. After thepreincubation with the various proteins, apoptosis was induced byaddition of staurosporine (Calbiochem, San Diego, Calif.) to a finalconcentration of 10 μM in the presence of the Apopercentage dye(Biocolor, Belfast, N. Ireland) diluted to a final concentration of 1/20of the provided stock solution per the manufacturer's instructions.

After 1 hour incubation at 37° C. in a humid atmosphere containing 5%CO₂ in air the cells were washed in phosphate buffered saline (PBS) and500 μl of DMEM high glucose without phenol red (Gibco, Grand Island,N.Y.) with 5% fetal bovine serum (Omega Scientific, Tarzana, Calif.),100 U/ml penicillin G sodium (Gibco), 100 mg/ml streptomycin sulphate(Gibco) and 2 mM glutamine (Gibco) added to the cells.

Cells were photographed immediately after washing, using a Zeiss IMinverted microscope connected to a Spot QE digital camera. An average of4 fields at 100× magnification were photographed per coverslip andnumbers of apoptotic cells were counted using the image analysissoftware Cell Counter (written by Dr. L. O. Mosnier, The ScrippsResearch Institute). For each experiment representative fields of thecells were photographed using phase contrast and the total number ofcells present was counted. The percentage of apoptosis is expressed asthe number of apoptotic cells relative to the total number of cells.Repeated control experiments were performed (MTT based assay, Celltiter96 Aqueous non-radioactive cell proliferation assay, Promega, Madison,Wis.) to ascertain that the cells did not become detached. In addition,on occasions when disruption of the confluent cell layer was observed,the data point was excluded from further analysis and repeated.

Clotting Assays

Dilute prothrombin time clotting assays were performed, as follows.Plasma (50 μL) was incubated with 50 μL of APC in HBS with 0.5% BSA atAPC concentrations from 8 to 32 nM (2.7-11 nM final concentration) for 3min at 37° C. Then clotting was initiated by adding 50 μL Innovin (DadeBehring Inc., Newark, Del.) diluted 1:60 in HBS, 0.5% BSA, 25 mM CaCl₂.The clotting time was measured using an ST4 coagulometer (DiagnosticaStago, Asnieres, France). For APTT clotting assays, 50 μL of plasma wasmixed with 50 μL of APTT reagent (Platelin L S, Organon Technika Corp,Durham, N.C.) and preincubated for at 37° C. for 3 minutes. Then 2 μLAPC was added followed by 50 μL of HBS, 0.5% BSA, 5 mM CaCl₂. Theclotting time was recorded using an ST4 coagulometer (Diagnostica Stago,Asnieres, France).

APC Inactivation

APC inactivation by serpins present in plasma was measured essentiallyaccording to the protocol of Heeb et al (J. Biol. Chem., 265:2365-2369,1990). Briefly, either human plasma or a mix of pure serpin inhibitors(PCI and/or α1-antitrypsin) was preincubated at 37° C., then APC wasadded. At selected times aliquots were removed and assayed for APCactivity with an APC specific chromogenic substrate.

Factor Va Inactivation

Inactivation of FVa was measured as follows. A mixture of 1 nM FVa with25 μM phospholipid vesicles was made in 50 mM HEPES, pH 7.4, 100 mMNaCl, 0.5% BSA, 5 mM CaCl₂, 0.1 mM MnCl₂. Inactivation was initiated bythe addition of APC. One microliter aliquots were removed at time pointsand added to 40 μL containing 1.25 nM factor Xa (FXa) with 31 μMphospholipid vesicles, followed by addition of 10 μL 3 μM prothrombin(final concentrations: 1 nM FXa, 20 μM FVa, 25 μM phospholipid vesiclesand 0.6 μM prothrombin). After 2.5 min a 15 μL aliquot of this mixturewas quenched by addition to 55 μL HBS containing 10 mM EDTA, 0.5% BSA,pH 8.2. Chromogenic substrate CBS 34-47 (Diagnostica Stago, Asnieres,France) was added and the rate of thrombin formation was assessed bymeasuring the change in absorbance at 405 nm. Curve fitting of thesepseudo-first order time courses of FVa inactivation was done accordingto Nicolaes et al. (supra, 1995) using equation 1:

$\begin{matrix}{{Va}_{t} = {{{Va}_{0} \cdot ^{{- {({k_{506} + k_{306}^{\prime}})}} \cdot t}} + {B \cdot {Va}_{0} \cdot \frac{k_{506} \cdot ^{({{- k_{306}} \cdot t})}}{\left( {k_{506} + k_{306}^{\prime} - k_{306}} \right)} \cdot \left( {1 - ^{{- {({k_{506} + k_{306}^{\prime} - k_{306}})}} \cdot t}} \right)}}} & {{equation}\mspace{14mu} 1}\end{matrix}$

Those skilled in the art will recognize other disease states and/orsymptoms, which might be treated and/or mitigated by the presentinvention. For example, the present invention may be used to treatmyocardial infarction, other heart diseases and their clinical symptoms,endothelial injury, adult respiratory distress syndrome (ARDS), andfailure of the liver, kidney, or central nervous system (CNS). There aremany other diseases which benefit from the methodologies of the presentinvention such as for example, coronary arterial occlusion, cardiacarrhythmias, congestive heart failure, cardiomyopathy, bronchitis,neurotrauma, graft/transplant rejection, myocarditis, diabeticneuropathy, and stroke. Life threatening local and remote tissue damageoccurs during surgery, trauma, and stroke when major vascular beds aredeprived for a time of oxygenation (ischemia) then restored with normalcirculation (reperfusion). Cell death and tissue damage can lead toorgan failure or decreased organ function. The compositions andmethodologies of the present invention are useful in treatment of suchinjury or prevention thereof.

In summary, two examples of the variants of recombinant APC mutants ofthis invention, namely KKK191-193AAA-APC and RR229/230AA-APC areprovided, that have substantial reductions in anticoagulant activity butthat retain normal or near-normal levels of anti-apoptotic activity. Theinvention encompasses APC variants such as these, which have the highlydesirable property of a high ratio of anti-apoptotic to anticoagulantactivity. The invention further encompasses variants having more modest,yet still beneficial, ratios of anti-apoptotic to anticoagulantactivity; such variants also would be expected to be cytoprotectivewhile having significantly reduced risk of bleeding. The invention isnot limited to variants of APC, but also includes protein C mutantswhich are capable of yielding desirable APC mutants, i.e., those thatwould have the same desirable activity ratios. The invention also is notlimited to mutations on loop 37, calcium loop, or autolysis loop; theinvention encompasses mutations of residues on other surface loops ofthe protease domain that produce the desired cytoprotective toanticoagulant ratio. Thus, APC and protein C variants of the inventionare expected to be useful for therapy for subjects who will benefit fromAPC protective activities that are independent of APC's anticoagulantactivity. Subjects would include patients at risk of damage fromapoptosis to blood vessels or tissue in various organs. Morespecifically, but not exclusively, these subjects will include, forexample, those suffering severe sepsis, ischemia/reperfusion injury,ischemic stroke, acute myocardial infarction, acute or chronicneurodegenerative diseases and organ transplantation, among otherconditions.

Example 5 Methods

This example includes refined data from Table 1 incorporating additionalexperiments that are averaged in and improved data analysis along withdata for the variant S360A-APC. Furthermore, the anticoagulant data wascollected using the APTT assay instead of the PT assay (as mentioned inTable 2). Therefore, this refined data is presented as Table 2. Thisexample also includes more detailed analysis of the amidolytic,anticoagulant and anti-apoptotic activities of the variants of APC(FIGS. 3-6). For this example, the following methods were employed.

Human alpha-thrombin was purchased from Enzyme Research Laboratories(South Bend, Ind.). Normal human citrate-anticoagulated plasma was fromGeorge King Bio-Medical, Inc. (Overland Park, Kans.). The chromogenicsubstrate L-Pyroglutamyl-L-prolyl-L-arginine-p-Nitroaniline hydrochoride(S-2366) was obtained from Chromogenix (Franklin, Ohio).

Preparation of Recombinant Activated Protein C

Mutant protein C expression vectors were constructed and recombinantprotein C mutants were purified from conditioned media as described(Gale et al., supra, 2002; Gale et al., supra, 1997). Purified protein Cwas activated by thrombin (Gale et al., supra, 2002; Gale et al., supra,1997). Briefly, Protein C in HBS (50 mM HEPES, 150 mM NaCl) with 2 mMEDTA and 0.5% BSA, pH 7.4, at a concentration of 600 μg/mi was incubatedfor 2.5 h with 12 μg/ml thrombin at 37° C. followed by the addition of1.1 units of hirudin per unit of thrombin to inactivate the thrombin.Subsequently, thrombin was removed by anion-exchange chromatography withNaCl gradient elution (Yan et al., Biotechnology, 8:655-661, 1990).Residual thrombin, as determined by fibrin clotting, accounted for lessthan 0.00025% (mol thrombin/mol APC) of the protein. Concentrations ofrwt-APC and APC mutants were determined by active-site titration adaptedfrom Chase and Shaw (Chase and Shaw, supra, 1967) using APC at ˜8 μM inHBS and p-nitrophenol-guanidino benzoate at 0.1 mM and using anextinction coefficient for p-nitrophenol of 11,400 M⁻¹cm⁻¹ (at pH 7.4)as described (Gale et al., supra, 2002). The concentration of S360A-APCwas determined by Asserachrom Protein C ELISA from American Bioproducts(Parsippany, N.J.) (Gale et al., supra, 1997).

APC Activity Assays

Amidolytic (S-2366) assays were performed as described (Gale et al.,supra, 2000; Gale et al., supra, 1997). APTT clotting time assays wereperformed according to the following procedure. Plasma (50 μl) wasincubated for 1 min with kaolin/cephalin (50 μl) (C.K. Prest 2,Diagnostica Stago, Parsippany, NJ) at 37° C., and then 25 μl APC in HBSwith 0.5% BSA was added at final APC concentrations from 0.5 nM-32 nMand incubated for an additional 3 min. Clotting was then initiated byadding 50 μl of 50 mM CaCl₂ in HBS and the clotting time was recordedusing an Amelung KC 4a micro coagulometer (Sigma Diagnostics, St Louis,Mo.).

APC's cytoprotective effects were determined in assays ofstaurosporine-induced endothelial cell (EA.hy926) apoptosis as described(Mosnier and Griffin, supra, 2003). APC (0.16-100 nM) was incubated withcells for 5 h prior to induction of apoptosis by staurosporine (10 μM, 1h) unless otherwise indicated, and apoptosis was assessed byApopercentage dye from Biocolor (Belfast, N. Ireland) which measuresphosphatidylserine translocation to the outside surface of the cellmembrane. Blocking antibodies against PAR-1 (WEDE-15 and ATAP-2 kindlyprovided by Dr L. Brass) and against EPCR (Zymed) were used as described(Mosnier and Griffin, supra, 2003). For activated caspase-3immunofluorescence staining and DAPI nuclear staining (5 μg/ml) ofstaurosporine-treated (2 μM, 4 h) EA.hy926 endothelial cells that hadbeen incubated with APC (25 nM, 5 h) prior to apoptosis induction, themanufacturer's instructions were followed using a rabbit anti-activatedcaspase-3 antibody (Promega) and Alexa-fluor-568 labeled secondary goatanti-rabbit (Molecular Probes).

PAR-1 Peptide Cleavage.

The interactions of rwt-APC and APC variants (500 nM) with PAR-1N-terminal tail peptide (TR33-62) were studied using a synthetic peptiderepresenting PAR-1 residues 33-62 (Bio Synthesis Inc., Lewisville,Tex.). The peptide sequence was A³³TNATLDPR⁴¹SFLLRNPNDKYEPFWEDEEKN⁶² andwas cleaved by APC between Arg41 and Ser42. The substrate peptide andthe two peptide products of thrombin or APC cleavage at Arg41 (TR3341and TR42-62) were resolved and analysed by reverse phase HPLC andquantified essentially as described (Arosio et al., Biochemistry,39:8095-8101, 2000). All TR33-62 cleavage experiments with APC contained5 nM hirudin to assure that the observed cleavage was not due tothrombin contamination.

Results

The anti-apoptotic, anticoagulant and amidolytic activities ofRR229/230AA-APC and KKK191-193-AAA-APC were determined and compared tothe activities of recombinant wild type (rwt)-APC and of ahydrolytically inactive mutant, S360A-APC, containing Ala in place ofthe active site residue, Ser360. The two APC protease domain loopvariants, RR229/230AA-APC and KKK191-193-AAA-APC, had the same enzymaticactivity against a small chromogenic substrate, S-2366, as recombinantwild-type APC (rwt-APC) (FIG. 3 a), indicating the structural andfunctional preservation of the APC active site, whereas these variantshad markedly decreased anticoagulant activity (FIG. 3 b) that was due toimpaired cleavage at Arg5O6 in factor Va (see Table 2).

TABLE 2 Recombinant wild type and mutant APC activities.* factor VaPAR-1 inactivation peptide cytoprotective anticoagulant cytoprotectivecleavage at amidolytic (TR33-62) APC sequence activity activity toanticoagulant Arg⁵⁰⁶ (Arg³⁰⁶) activity cleavage Mutant (mutated to Ala)(% rwt-APC)^(†) (% rwt-APC)^(‡) ratio^($) (% rwt-APC)

(% rwt-APC)^(¶) (% rwt-APC)^(#) rwt-APC* none 100% 100% 1.0 100% (100%)100% 100% 229/230-APC 227-DL RR E-232  94%  13% 7.2 25% (110%) 102% 116%3K3A-APC 189-DS KKK A-195 114%  4.6% 25 11% (67%) 109%  88% S360A-APC358-GD S G-362   <1%**   23%^(††) 0 <1%** (<1%**)   <1%**   <3%**(anticoagulant activity determined by APTT) *Recombinant wild-type APC(rwt-APC) activity was defined as 100% and values for mutant APC's aregiven as percentage of rwt-APC activity. ^(†)Derived from theconcentrations of APC required for half-maximal inhibition of thestaurosporine-induced apoptosis (FIG. 2a). ^(‡)Based on the APTTdose-response data determined for rwt-APC and APC variants (0.5 nM-32nM) (FIG. 1b). ^($)Derived from the ratio of relative activities forcytoprotective and anticoagulant activities given in the previous twocolumns of this Table.

Based on apparent second-order rate constants determined previously(Gale et al., supra, 2002; Gale et al., supra, 1997). ^(¶)Based on theamidolytic activity determined for rwt-APC and APC variants (0.5 nM-32nM) (FIG. 1a). ^(#)Based on the catalytic efficiency derived from FIG. 4for cleavage of the PAR-1 peptide (TR33-62) by rwt-APC and APC variants(500 nM). **No detectable activity under the conditions of the assay.^(††)Anticoagulant activity of S360A-APC is not due to proteolysis offactor Va and in contrast to rwt-APC is independent of the incubationtime of APC with the plasma (Gale et al., supra, 1997).

Although S360A-APC had no chromogenic activity (FIG. 3 a), theanticoagulant activity of S360A-APC was ˜23% in the conditions of theAPTT assay (FIG. 3 b). As previously described, in contrast to normalrwt-APC, this anticoagulant activity is independent of the incubationtime of APC with plasma (Gale et al., supra, 1997) and appears toinvolve binding of APC exosites to factor Va such that there isinhibition of factor Xa and prothrombin binding to factor Va.

To determine cytoprotective activity of these APC variants,staurosporine-induced endothelial cell apoptosis (Joyce et al., supra,2001; Mosnier and Griffin, supra, 2003) was studied. APC-mediatedinhibition of staurosporine-induced apoptosis is time-dependent anddose-dependent and it requires APC's active site, PAR-1 and EPCR(Mosnier and Griffin, supra, 2003). Half-maximum inhibition ofstaurosporine-induced apoptosis by rwt-APC was achieved at 0.16 nM underthe conditions employed (FIG. 4 a). Dose-dependent inhibition ofapoptosis by RR229/230AA-APC and KKK191-193AAA-APC was indistinguishablefrom that of rwt-APC with half-maximum inhibition at 0.17 nM and 0.14nM, respectively (FIG. 4 a). No inhibition of apoptosis by an APC mutantlacking the active site serine, S360A-APC (Gale et al., supra, 1997),was observed (FIG. 4 a-c). The ability of rwt-APC and APC variants toinhibit generation of activated caspase 3 in endothelial cells exposedto staurosporine was monitored immunohistochemically. rwt-APC and thevariants, RR229/230AA-APC and KKK191-193AAA-APC, each similarly reducedactivated caspase-3-positive cells by approximately 70%, whereas theactive site mutant, S360A-APC, had no effect (FIG. 4 b-c). Thus, certainprotease domain residues essential for normal anticoagulant activity ofAPC, namely Arg229 and Arg230 and Lys191, Lys192 and Lys193, are notrequired for normal anti-apoptotic activity of APC.

APC anti-apoptotic effects require PAR-1 and EPCR (Cheng et al., supra,2003; Mosnier and Griffin, supra, 2003). Similarly, the anti-apoptoticactivity of RR229/230AA-APC and KKK191-193AAA-APC in assays ofstaurosporine-induced endothelial cell apoptosis required PAR-1 and EPCRbecause the cytoprotective activity of each APC variant was inhibited by72% and 69% in the presence of antibodies against EPCR that blockbinding of APC to the receptor and by 88% and 55% in the presence ofblocking anti-PAR-1 antibodies, respectively (FIG. 5). These resultsindicate that interactions between cells and the two APC variants, likerwt-APC, require PAR-1 and EPCR.

Cleavage of Synthetic PAR-1 N-Terminal Tail by Wild Type and VariantAPC's

The absence of anti-apoptotic activity of S360A-APC and the requirementfor PAR-1 imply that a primary mechanistic step for APC's anti-apoptoticactivity involves PAR-1 proteolytic activation (Cheng et al., supra,2003; Mosnier and Griffin, supra, 2003). To characterize the effects ofthe mutations in APC on proteolytic activation of PAR-1 due to cleavageat Arg41, a synthetic 30-mer peptide representing the PAR-1 N-terminalsequence (residues 33-62 (TR33-62)) was studied as an APC substrate.This TR33-62 PAR-1 peptide is cleaved at Arg41 by thrombin (Arosio etal., supra, 2000). APC cleaves another PAR-1 synthetic N-terminalpeptide at Arg41, the thrombin cleavage site (Kuliopulos et al., supra,1999). Using HPLC quantitative analysis, we found that rwt-APC cleavedthe TR33-62 peptide at Arg41 and generated similar fragments asthrombin, TR33-41 and TR42-62, but at approximately a 25,000-fold lowercatalytic efficiency based on comparison of k_(cat)/K_(m) for the twoenzymes (data not shown). When the time course for TR33-62 cleavage wasmonitored using HPLC to quantify the disappearance of the peak for theTR33-62 substrate and the appearance of the TR42-62 product, the resultsshowed that there were no substantial differences in the rate of TR33-62cleavage between the rwt-APC, RR229/230AA-APC and KKK191-193AAA-APC(FIG. 6). Similarly, no significant differences in APC-inducedCa⁺⁺-intracellular flux monitored as FURA-2-AM fluorescence changes wereobserved in EA.hy926 endothelial cells when rwt-APC was compared withthe two anti-apoptotic APC variants, RR229/230AA-APC andKKK191-193AAA-APC (data not shown). These results are consistent withthe hypothesis that APC cleaves PAR-1 at Arg41 and that the mutations inthe two APC variants described here with reduced anticoagulant activitybut with normal anti-apoptotic activity did not significantly reduce theability of the protease domain of APC to cleave PAR-1 at Arg41.

In summary, to generate recombinant APC variants with reduced risk ofbleeding due to reduced anticoagulant activity, we dissected APC'santicoagulant activity from its cytoprotective activity by site-directedmutagenesis. Using staurosporine-induced endothelial cell apoptosisassays, we show here that Ala mutations (RR229/230AA and KKK191-193AAA)in two APC surface loops that severely reduce anticoagulant activityresult in two APC variants that retain normal anti-apoptotic activitythat requires protease activated receptor-1 and endothelial cell proteinC receptor. Moreover, these two APC variants retain a normal ability tocleave a PAR-1 N-terminal peptide at Arg41. To compare these two APCvariants to rwt-APC in terms of their relative anti-apoptotic andanticoagulant activities (determined by APTT; note in table 1anticoagulant activity was determined by dilute PT), we assigned theobserved activity of rwt-APC a value of 100% and calculated the percentactivity of each APC variant from dose-response data (FIGS. 3 and 4).This normalization inherently yields a “cytoprotective to anticoagulant”ratio for rwt-APC of 1.0 (Table 2). When the ratio of anti-apoptoticactivity to anticoagulant activity was calculated for the APC mutants(Table 2), the two APC variants exhibited 7-times and 25-times greateranti-apoptotic activity relative to anticoagulant activity compared torwt-APC, respectively. These ratios are similar to the values seen inTable 1 calculated using the dilute PT assay for anticoagulant activity.

Thus, these data imply that the RR229/230AA and KKK191-193AAA mutationsin APC which cause decreased cleavage at Arg506 in factor Va do notimpair cleavage at Arg4l in PAR-1.

The references and patents cited herein, are hereby incorporated byreference in their entirety.

1. A method of selecting potentially therapeutic cytoprotective variantsof recombinant activated protein C, said activated protein C having aprotease domain, comprising: mutating a recombinant activated protein Cat a surface loop of said protease domain to make an activated protein Cvariant; measuring the anticoagulant activity of said activated proteinC variant; measuring the anti-apoptotic activity of said activatedprotein C variant; calculating the ratio of anti-apoptotic activity toanti-coagulant activity; and identifying the activated protein C variantas potentially therapeutic if said ratio is greater than 1.0.
 2. Themethod of claim 1, wherein the ratio is greater than about
 2. 3. Themethod of claim 1, wherein the ratio is greater than about
 4. 4. Themethod of claim 1, wherein the ratio is greater than about
 8. 5. Themethod of claim 1, wherein said surface loop is selected from the groupconsisting of loop 37, calcium loop, and autolysis loop.
 6. A method ofselecting potentially therapeutic cytoprotective variants of recombinantprotein C, said protein C having a protease domain, comprising: mutatinga recombinant protein C at a surface loop of said protease domain tomake a protein C variant; converting said protein C variant to anactivated protein C variant; measuring the anticoagulant activity ofsaid activated protein C variant; measuring the anti-apoptotic activityof said activated protein C variant; calculating the ratio ofanti-apoptotic activity to anti-coagulant activity; and identifying theprotein C variant as potentially therapeutic if said ratio is greaterthan 1.0.
 7. The method of claim 6, wherein the ratio is greater thanabout
 2. 8. The method of claim 6, wherein the ratio is greater thanabout
 4. 9. The method of claim 6, wherein the ratio is greater thanabout
 8. 10. The method of claim 6, wherein said surface loop isselected from the group consisting of loop 37, calcium loop, andautolysis loop.
 11. A method of selecting potentially therapeuticcytoprotective variants of recombinant activated protein C, comprising:(a) providing a library of candidate agents which are protein Cvariants, wherein each of said protein C variants has a protease domain,wherein each of said protein C variants comprises at least one mutationat a residue in a surface loop of said protease domain; (b) convertingeach of said protein C variants to activated protein C variants; (c)determining anti-apoptotic activity of said activated protein C variantsin one or more stressed or injured cells by exposing said cells to anapoptotic-inducing concentration of staurosporine in the presence of anamount of a candidate agent; (d) determining anticoagulant activity ofsaid candidate agents that are assayed in (c) by performing a diluteprothrombin time clotting assay; (e) calculating the ratio of theanti-apoptotic activity determined in (c) to the anticoagulant activityof (d); and (f) selecting candidate agents having ananti-apoptotic:anticoagulant activity ratio greater than 1.0.
 12. Themethod of claim 11, wherein said ratio is greater than about
 2. 13. Themethod of claim 11, wherein said ratio is greater than about
 4. 14. Themethod of claim 11, wherein said ratio is greater than about
 8. 15. Themethod of claim 11, wherein said surface loop is selected from the groupconsisting of loop 37, calcium loop, and autolysis loop.
 16. A method ofselecting potentially therapeutic cytoprotective variants of recombinantactivated protein C, comprising: (a) providing a library of candidateagents which are activated protein C variants, wherein each of saidactivated protein C variants has a protease domain, wherein each of saidactivated protein C variants comprises at least one mutation at aresidue in a surface loop of said protease domain; (b) determininganti-apoptotic activity of said activated protein C variants in one ormore stressed or injured cells by exposing said cells to anapoptotic-inducing concentration of staurosporine in the presence of anamount of a candidate agent; (c) determining anticoagulant activity ofsaid candidate agents that are assayed in (b) by performing a diluteprothrombin time clotting assay; (d) calculating the ratio of theanti-apoptotic activity determined in (b) to the anticoagulant activityof (c); and (e) selecting candidate agents having ananti-apoptotic:anticoagulant activity ratio greater than 1.0.
 17. Themethod of claim 16, wherein said ratio is greater than about
 2. 18. Themethod of claim 16, wherein said ratio is greater than about
 4. 19. Themethod of claim 16, wherein said ratio is greater than about
 8. 20. Themethod of claim 16, wherein said surface loop is selected from the groupconsisting of loop 37, calcium loop, and autolysis loop
 21. An agentselected by the method of claim
 1. 22. An agent selected by the methodof claim
 6. 23. An agent selected by the method of claim
 11. 24. Anagent selected by the method of claim 16.